In the 70's, the advent of optical and impedance-based measurements permitted for the first time relatively rapid automated sizing, counting, and allied investigations of individual cells. Impedance-based systems, in which cells were caused to proceed through an orifice, are based on the proposition that certain cell parameters, principally related to size, correlate with changes of impedance at the orifice as cells pass therethrough. Competing with the impedance systems were early optical flow cytometry systems, in which hydrodynamic principles were utilized to pass cells rapidly, substantially one at a time, through a zone of focused illumination such as that from a laser beam. Each cell interacts characteristically with the beam, and depending variously on light extinction, narrow and wide angle scanner, and back scatter (i.e., reflection), computation algorithms allowed for substantial discrimination among cell types based on these optical interaction parameters. See, for example, U.S. Pat. Nos. 3,662,176 to Kamentsky et al., 3,785,735 to Friedman et al., and 3,705,771 to Friedman et al.
After a time, principles of optical flow cytometry were adapted to ever-increasingly sophisticated approaches to analysis. Dyes such as acridine orange, which fluoresce in the presence of certain types of illumination, were found to be taken up in different proportions by different types of cells depending on their pathological and morphological character. See, for example, U.S. Pat. No. 3,684,377 to Adams et al.
Thus, while impedance-based and optical flow cytometry systems competed vigorously for predominance in the clinical marketplace, the optical systems found favor and acceptance in research laboratories, wherein relatively high instrument costs, and the associated elaborate processing and preprocessing steps were easily outweighed by the powerful and extensive information which could be learned on a cell-by-cell basis. In fact, several major manufacturers came to offer elaborate, powerful optical flow cytometry systems directed to the research laboratory marketplace, in which plural lasers were utilized in conjunction with a variety of sample processing and dyeing techniques, and wherein elaborate algorithms allowed the basic optical interaction parameters (i.e. extinction, back scatter, narrow angle scatter, wide angle scatter, and plural color fluorescence) to assemble highly accurate data regarding subpopulations of cells in the sample. Prime examples of these types of instruments are those offered under the brand name CYTOFLUOROGRAF.RTM. by Ortho Diagnostic Systems Inc., Raritan, N.J., and its predecessors in interest.
Optical flow cytometry instruments therefore enjoyed an important role in the development of modern immunology and immunologically based reagents such as those utilizing monoclonal and polyclonal antibodies. Earlier immunologic experiments were premised on elaborate incubation and manual counting and analysis techniques, as well as manual achievement of physical isolation of respective subclasses. Along the way, however, it was determined that optical flow cytometry techniques could vastly attenuate the duration of such experiments by providing analysis and sorting on an automated basis. Thus, the very development of monoclonal antibodies and the like reagents occurred hand in hand with the continuing refinement of optical flow cytometry techniques and apparatus. Moreover, as the various antibody secreting hybridoma cell lines were developed, and their characteristic antibodies became available, the optical flow cytometry systems became a modality of choice for use of the antibodies for diagnostic, monitoring, and the like purposes. For example, Ortho Pharmaceutical and Ortho Diagnostic Systems Inc., both of Raritan, N.J., have developed a series of monoclonal reagents under the "OKT".RTM. trademark, which react selectively with human T-lymphocyte cells. These monoclonal antibodies are capable of carrying markers which fluoresce in the presence of select illumination, and hence offer the capability of cellular analysis and sorting in optical flow cytometry systems. U.S. Pat. No. 4,284,412 to Hansen et al. describes and claims methods and apparatus for automated identification and enumeration of blood cell subclasses, such as human T-cell subclasses, through the use of optical flow cytometry systems. The assignee hereof, which is also the assignee of the Hansen et al. patent, offers commercially a line of systems under the SPECTRUM trademark which is designed in part for these immunologic techniques. Other manufacturers as well, have commercially offered antibodies and instruments which they claimed are the equivalents of Ortho's reagents and systems.
Thus, the sciences of immunology and cell analysis instrumentation have been developed in a partnership, without which it is doubtful that either would have advanced to its present state. The associated difficulty, however, is that the optical flow systems are relatively expensive, and their elaborate optical and hydrodynamic systems are mechanically complex and sensitive, and therefore tend to be service intensive. Thus, while there is a strong need to move immunologic analyses to everyday use in the clinical laboratory, progress tends to be impeded by the overall system cost and the high level of effort required to maintain system accuracy and reliability. Further, there is a standing need to have test formats and procedures which require minimal operator sophistication, which object is not always met in modern optical flow cytometry immunologic testing techniques.
It is, accordingly, a primary object of the present invention to provide apparatus and techniques which improve upon and simplify present optical techniques for immunology and hematology. It is a further object to provide instruments for such analysis which are low in cost, high in reliability, and which require minimal sophistication on the part of the operator.